Improved orange and red Ca²⁺ indicators and photophysical considerations for optogenetic applications

We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca²⁺) indicators to include orange and improved red fluorescent variants, and validated the latter for combined use with optogenetic activation by channelrhodopsin-2 (ChR2). These indicators feature intensiometric signal changes that are 1.7- to 9.7-fold improved relatively to the progenitor Ca²⁺ indicator, R-GECO1. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca²⁺ indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca²⁺ imaging traces during optogenetic activation with ChR2. We demonstrate, in both a beta cell line and slice culture of developing mouse neocortex, that these artifacts can be avoided by using an appropriately low intensity of blue light for ChR2 activation.